Nanometer-sized proteins generate micron-sized tags at distinct cellular sites to spatially regulate function across biology. Typically, such tags depend on proteins specifically recognizing structural features (e.g. DNA sequence at telomeres) that remain stable for periods that are much longer than protein association and activity timescales. However, it is unclear how proteins can selectively tag microtubules, which turnover on timescales faster than that of typical biochemical mechanisms (e.g. phosphorylation). To address this long-standing question, we focused on PRC1 and kinesin-4, two microtubule-associated proteins required to assemble the spindle midzone, a specialized array of microtubule bundles that keeps segregated chromosomes apart and positions the division plane in human cells...Read More

Examining dynein functions in these diverse dynamic processes has been difficult using conventional approaches. In this report we describe the discovery of ciliobrevins, dihydroquinazolinone-based small molecules that are the first selective inhibitors of cytoplasmic dynein. We show that ciliobrevin treatment disrupts protein trafficking within the primary cilium and leads to shorter and malformed cilia. Consistent with these effects, Hedgehog signaling is blocked by ciliobrevins. These compounds also inhibit proper mitotic spindle assembly and block organelle transport in cultured cells...Read More

The metaphase spindle must generate nanonewton-scale forces to pull chromosomes apart and must also be robust to withstand opposing forces of equal magnitude. Forces also act on the metaphase spindle to control its position and size. In addition, chromosomes, which are proportionately large cargoes, must be allowed to move through the spindle’s dense cytoskeletal polymer network. The forces associated with these chromosome motions exert deformations within the metaphase spindle. How this essential cellular structure maintains functional fidelity and structural stability in the face of these forces that can vary in magnitude, orientation and time-scales is poorly understood. To address this question we measured the orientation- and timescale-dependent micromechanical properties of the metaphase spindle...﻿Read More﻿

This study represents an important step in our efforts to reconstitute a ‘minimal mitotic spindle’ with purified proteins. The biophysical and structural characterization of the key non-motor microtubule-associated proteins (MAPs) required for cell division lags far behind that of motor proteins (e.g. kinesins). To fill this knowledge gap and to characterize the proteins needed for our reconstitution assays, we focused on PRC1. This non-motor MAP, or its homologs, is needed for normal cell growth in plants and fungi. In metazoans, PRC1 is needed for successful cell division. We combined X-ray crystallography, electron microscopy, and TIRF (total internal reflection fluorescence) microscopy approaches to analyze PRC1’s functions...﻿Read More﻿

Error-free cell division depends on spatial and temporal cues regulating micron-scale organization. In current models, substrate phosphorylation plays a central role in generating these regulatory cues. While the kinase and phosphatase localizations during cell division have been extensively analyzed, the dynamics of phosphorylation remain poorly characterized. To fill this gap in our knowledge, we developed FRET-based sensors to examine in dividing cells the substrate phosphorylation dynamics that depend on Aurora kinase, a conserved cell cycle regulator in eukaryotes and an anti-cancer drug target...Read More